Novel haptene steroids

ABSTRACT

Novel steroids of the formula   WHEREIN X is selected from the group consisting of O and,   Y is selected from the group consisting of hydrogen and -OH and when R1 is -(CH2)a-COOH in which a is equal to 2n + 3 and n is 0, 1 or 2, R2 is   and when R1 is hydrogen, R2 is selected from the group consisting of AND N-Z-(CH2)c-COOH, b is a number from 3 to 18, c is a number from 1 to 8 and Z is selected from the group consisting of -Oand -NHCONH- which are haptenes or compounds capable of producing antibodies in vitro but are derived of antigenic properties and their preparation.

United States Patent 1191 Torelli et al.

[ Nov. 25, 1975 NOVEL HAPTENE STEROIDS [75] Inventors: Vesperto Torelli, Maisons-Alfort;

Andre Pierdet, Noisy-le-Sec, both of France [73] Assignee: Roussel-UCLAF, Paris, France [22] Filed: June 17, 1974 [211 App]. No.: 479,889

[30] Foreign Application Priority Data June 18. 1973 France 73.22114 [52] US. CL... 260/397.1; 195/51 F; 260/239.55 R; 260/3406; 260/340.9; 260/348 A; 260/397.4; 260/397.45; 260/3975 A; 424/88 [51] Int. Cl. C07J 1/00 [58] Field of Search 260/397.1

[56] References Cited UNITED STATES PATENTS 3,211,756 10/1965 Mazur 260/397.l

FOREIGN PATENTS OR APPLICATIONS 3.822.877 10/1963 Japan 260/397.1

Primary Examiner-Ethel G. Love Attorney, Agent, or Firm-Hammond & Littell [57 ABSTRACT Novel steroids of the formula wherein X is selected from the group consisting of =0 and,

and =N-Z-(CH ),.COOH. b is a number from 3 to 18, c is a number from 1 to 8 and Z is selected from the group consisting of O and NHCONH which are haptenes or compounds capable of producing antibodies in vitro but are derived of antigenic properties and their preparation.

9 Claims, No Drawings NOVEL HAPTENE STEIOIDS STATE OF THE ART C.R. Series C., vol. 276 I973 p. 303 describes haptenes which are derived from A -androstene-3B-oll7-one but the compounds of the invention are derivatives of estrone, estradiol and estratriol.

OBJECTS OF THE INVENTION It is an object of the invention to provide the novel A- ""-estratrienes of formula I.

It is another object of the invention to provide novel processes for the preparation of the steroids of formula I.

It is a further object of the invention to provide novel antigens and a process for their preparation.

These and other objects and advantages of the invention will become obvious from the following detailed description.

THE INVENTION The novel A" "-estratrienes of the invention have the formula wherein X is selected from the group consisting of=O and Y is selected from the group consisting of hydrogen and OH and when R, is (CI-I ),,--COOH in which a is equal to 2n 3 and n is 0, l or 2, R is and when R is hydrogen, R is selected from the group consisting of Amm -coon \H and R is -(CH ),,COOH where a has the above definition. Examples of these specific haptenes are A" -estratriene-3,l7B-diol-l lB-butyric acid and A" -estratriene-3-ol-l 7-one-l lB-butyric acid.

Another preferred group of haptenes of formula I are those where R is hydrogen and R is where b has the above definition. Specific compounds of this group are A-"' ""-estratriene-3,l7B-diol-7abutyric acid. A'"' "-estratriene-3-oll 7-one-7abutyric acid, A" -estratriene-3,l6a,]7B-triol-7abutyric acid and A" "-estratriene-3.l7B-diol-7aundecanoic acid.

A third preferred group of compounds of formula I are those where R, is hydrogen and R is =N- Z(CH ),.COOH and c and Z has the above definitions. Examples of these compounds are 7-carboxymethoxyimino-A" -estratriene-3. l 7B-diol. 7-carboxymethoxyimino-A'- -estratriene-3-ol-l 7-one and 4-carboxymethyl-2-semicarbazone of A"- -estraand =NZ(CH COOH. b is anumber from 3 to l8, 0 is a number from 1 to 8 and Z is selected from the group consisting of O and -NHCONH-.

triene-3, l 7B-diol-7-one.

The novel process of the invention for the preparation of the compounds of formula I in which R is (CH ),,-COOH and R is comprises reacting a compound of the formula (prepared as in French Pat. No. 1,497.593) wherein K and K are blocked keto in the form of a ketal and L is acyl of an organic carboxylic acid with an epoxidation agent followed by reaction with a saponification agent to form a compound of the formula III reacting the latter with an allyl magnesium halide to form a compound of the formula CH=CH reacting the latter with a deketalization agent and then a cyclization agent to obtain a compound of the formula reacting the latter with an aromatization agent to form compound of the formula reacting the latter with an acylating agent to form a compound of the formula VII wherein L is an acyl of an organic carboxylic acid subjecting the latter to ozonolysis to form a compound of the formula reacting the latter with an alkyl dialkylphosphonoacylate of the formula i i PCH- (CH=CH )"COOR R wherein R,, R and R are the same or different alkyls of l to 8 carbon atoms and n has the abovedefinit-ion followed by reaction with a saponificationagent toobtain a compound of the formula 4300a uctw &2 OH

, and reacting the latter with a hydrogenation agent1toobtain the compound of formula I wherein R, is t(CH- 2)(1COOH, a is equal to 2n 3, X is Y is hydrogen and R is The said product may be oxidized with an oxidation agent to form the corresponding compound of formula I wherein X is =0. V

The substituents K and K' may be the same or different and are preferably cyclic alkylene ketals ofi2 to 4 carbon atoms such as ethylene ketal or propyleneketal:

or dialkylketals such as dimethylketal or diethylketal.

L is preferably derived from a saturated or unsaturated aliphatic or cycloaliphatic carboxylic acid of l'to 6 18 carbon atoms. Examples of suitable acids are alka- LII noic acids such as formic acid, acetic acid, butyric acid, propionic acid, isobutyric acid or undecylic acid; cy- I cloalkylcarboxylic acids and cycloalkylalkanoic acids such as cyclopropylcarboxylic acid, cyclopentylcarboxylic acid, cyclohexylcarboxylic acid, cyclopentylacetic acid, cyclopentylpropionic acid, cyclohexylacetic acid or cyclohexylpropionic acids; benzoic acid; phenylalkanoic acids such as phenylacetic acid or phenylpropionic acid; or amino acids such as diethylaminoacetic acid or aspartic acid.

The epoxidation agent is preferably a peracid such as peracetic acid, perphthalic acid or m-chloroperbenzoic acid and the saponification agent is preferably an alkali metal base such as sodium hydroxide, potassium hydroxide, sodium amide, potassium tert.-butylate or lithium acetylide in ethylenediamine and the saponification reaction is preferably effected in a lower alkanol such as methanol or ethanol. The allyl magnesium halide is preferably the chloride or bromide.

The deketalization agent is preferably an acid agent such as hydrochloric acid, sulfuric acid, acetic acid, citric acid or p-toluene sulfonic acid and the reaction is preferably effected in at least one organic solvent such as alkanols like methanol, ethanol or isopropanol, a ketone such as acetone or a hydrocarbon such as benzene or toluene.

The cyclization agent may be a basic or acidic agent but is preferably basic such as an alkali metal alcoholate like sodium methylate, sodium ethylate or sodium or potassium tert.-butylate. The aromatization agent is preferably an acyl halide such as acetylhalide, preferably acetyl bromide, or acetic acid anhydride and the said reaction is followed by saponification with one of the agents discussed above. The esterification is preferably effected with the acid or a functional derivative thereof such as the acid halide, i.e. chloride or bromide, or its anhydride. L is preferably the same as L.

The ozonolysis is effected with ozone at temperatures of 50 to lO0C and the alkyl dialkylphosphonoacylate has the formula where preferably R and R are the same and are alkyl of l to 6 carbon atoms and R is alkyl of l to 6 carbon atoms.

The saponification agent is preferably an alkali metal base such as sodium hydroxide or potassium hydroxide, sodium amide, potassium tert.-butylate or lithium acetylide in ethylenediamine. The hydrogenation agent is preferably hydrogen in the presence of a catalyst such as palladium. The oxidation agent is preferably chromic anhydride, silver carbonate, silver silicate or lead tetraacetate.

In the most preferred mode of the invention, the epoxidation agent is perphthalic acid, the ally] magnesium halide is the bromide, the deketalization agent is sulfuric acid, the cyclization agent is sodium hydroxide, the aromatization agent is acetic acid anhydride, the saponification agent after aromatization is sodium hydroxide, the esterification agent is benzoyl chloride, the alkyl dialkylphosphonoacylate is methyl diethylphosphonoacetate, the hydrogenation agent is hydrogen in the presence of palladized carbon, the last saponification agent is potassium hydroxide and the oxidation agent is chromic anhydride.

The process of the invention for the preparation of compounds of formula I wherein R is hydrogen and R is and b has the above definition comprises reacting a steroid of the formula XII XIII

(CH CH OAc reacting the latter with a saponification agent to obtain a compound of the formula XIV EcH cH oH reacting the latter with an oxidation agent to form a compound of the formula and reacting the latter with a dehydrogenation agent to form a compound of the formula mp -c XVI which is a compound of formula I wherein R is hydrogen, X is =0, Y is hydrogen and R is and h is 3 to 187 The latter compound may be reacted with a reducing agent to form the corresponding compound of formula I wherein X is or may be reacted with an alcohol of the formula TOH where T is lower alkyl or a functional derivative thereof to form a compound of the formula (Nap/o XVII (CH COOT reacting the latter with an acid of the formula VOH where V is acyl of an organic carboxylic acid or a functional derivative thereof to form a compound of the formula OV i XVIII (CH COOT reacting the latter with an epoxidation agent to form a compound of the formula XIX subjecting the latter to hydroylsis to form a compound of the formula and reacting the latter with a reducing agent thenwith a saponification agent to form a compound of formula l where R is hydrogen, X is Y is OH, R is (CH2 ),,COOH

andbis3tol8. L hasthe same preferred values indicated above and Hal is chlorine or bromine and M is preferably benzyl or tetrahydropyranyl. The acid agentlis preferably hy-,

drochloric acid, sulfuric acid, citric acid, acetic acidlor p-toluene sulfonic acid and the esterification agentis the acid AcOH or a functional derivative thereof and Ac has preferably the same values as L above.

The saponification agent islpreferably one, of those discussed above such as sodium hydroxide, potassium hydroxide, sodium amide, potassium tert.-butylate or lithium acetylide in ethylenediamine and the reaction is effected in a lower alkanol such as methanol or ethaQ, nol. The oxidation agent may be one of thosepreviously mentioned such as chromic anhydride and the dehydrogenation is preferably effected biochemically,

such as with Arthrobacter Simplex UC lO47.'The re-@ ducing agent is preferably hydrogen in the presence of a palladium catalyst.

The esterification agent for the compound of formula XVI is preferably the alcohol, TOH where T is alkyl of l to 8 carbon atoms. The agent toesterify the 3- hydroxy and the enolic form of the l7'keto group, is preferably an acid anhydride such as or a derivative of the formula I wherein R is acyl of an organic 'carboxylic acid of l to 8, preferably 1 to 4 carbon atoms. The epoxidation agent is preferably a peracid such as perchloric' acid,

perphthalic acid or hexafluoroacetone hydroperoxide.

The hydrolysis is effected in an acid media such as sulfuric acid or acetic acid and the reducing agent is preferably a mixed hydr de such as sodium or lithium boroacetic anhydride 4 hydride. The saponificati on agent may be one of those discussed above.

In the most preferred mode of this process, L is acetyl, Hal is chlorine, and M is tetrahydropyranyl. The acid agent is hydrochloric acid, the esterification agent is acetic anhydride, the saponification agent is methanolic potassium hydroxide, the reducing agent is sodium borohydride, the esterification agent of the acid function is ethanol, the esterification agent of 3- hydroxy and of the enolic form of the 17-keto group is isopropenyl acetate, the epoxidation agent is hexafluoroacetone hydroperoxide, the hydrolysis reaction is effected in a sulfuric acid medium, the reducing agent is sodium borohydride and the last saponification agent is methanolic potassium hydroxide.

The process of the invention for the preparation'of compounds of formula 1 wherein'R is hydrogen and R is =NZ(CH -COOH wherein Z and c have the above definitions comprises reacting a compound of the formula XXI with a compound of the formula H2NZ(CH2).-COOH to form the corresponding compound of formula XXI I which is a compound of formula 1 wherein R is hydrogen, X is Y is hydrogen, R is NZ--(CH COOH, Z is selected from the group consisting of -O and NH- CONH, and C is a number from 1 to 8.

which can then be reacted with an oxidizing agent to a compound of the formula which is a compound of formula I wherein R is hydrogen, X is oxygen, Y is hydrogen, R is NZ (CH ),.COOH, Z is selected from the group consisting of -O and NHCONH-, and C is a number from 1 to 8.

In a preferred mode of the invention, the compound of the formula H NZ-(CH COOH is used in the form of its acid addition salt such as its hydrochloride and the oxidation agent may be chromic anhydride. The 7-keto estradiol of formula XXI is described in U.S. Pat. No. 2,418,603.

The compounds of formula I are haptenes useful for the preparation of antigens by forming a mixed acid anhydride thereof with an lower alkyl haloformate. particularly isobutyl chloroformate and reacting the resulting mixed anhydride with beef serum albumin. From these antigens, antibodies may be prepared by known methods such as described by Er1anger[.1. Biol. Chem., Vol. 228, p. 713].

The antibodies obtained are specific for the starting steriods which are estrone, estradiol and estriol which is evidenced by classical methods, particularly dialysis to equilibrium. This specificity of the antibodies obtained makes them usable as agents at the dosage used for estrone, estradiol or estriol. They are of interest for the dosing power for regulating the level of estriol in the blood and urine of pregnant women to permit the detection of certain anomalies of the fetus.

1n the following examples there are described several preferred embodiments to illustrate the invention. However. it should be understood that the invention is not intended to be limited to the specific embodiments.

EXAMPLE 1 A" -estratriene-3,17B-diol-1 1B-butyric acid STEP A: Bis 3,3 5,5-ethylenedioxy-9a,11a-epoxy-4.5- secoestrane-17B-o1 22 g of monoperphthalic acid were added at 20C with stirring to a mixture of 21.9 g of the benzoate of bis-3,3 5,5-ethy1enedioxy-4,S-seco-A "-estrene-17B- ol (prepared as described .in French Pat. No. 1,497,593) in 220 ml of tetrahydrofuran and the mixture was allowed to stand for about 16 hours. Then. the

' mixture was poured into a saturated sodium bicarbonate solution and the mixture was vacuum filtered. The precipitate was washed to obtain 22.2 g of a product melting at 160C which was used as is. 31.98 g of the said product were added to 130 ml of methanol and the suspension was added to 130 m1 of 1.74 N methanolic potassium hydroxide. The mixture was refluxed for 1 hour, was cooled, diluted with water and was extracted with methylene chloride. The extracts were dried and evaporated to dryness to obtain 23.6 g of resin which was crystallized from isopropyl ether to obtain 22.3 g of bis 3,3 5,5 -ethy1enedioxy-9a, 1 la-epoxy-4,5-secoestrane-17B-ol melting at 148C. STEP B: Bis 3,3 5,5-ethylenedioxy-11B-al1yl-4.5-secoestrane-9a, 17B-dio1 32.4 g of bis 3,3 5,5-ethylenedioxy-9a, Ila-epoxy- 4,5-seco-estrane-l7B-ol were added with stirring under a nitrogen atmosphere to 1 m1 of a 0.65 M solution of allyl magnesium bromide in tetrahydrofuran and the solution was heated to 60C for 4% hours. The mixture was cooled and stood overnight at room temperature under a nitrogen atmosphere. After cooling the mixture to C, 45 ml of methanol and 100 ml of ether were added thereto slowly and then ml of water were added. The reaction mixture was poured into 1000 ml of a saturated ammonium chloride solution and the aqueous phase was decanted and extracted with ether. The extracts were dried and distilled to dryness under reduced pressure to obtain 45 g ofa crystalline product which was empasted with a mixture of ether and essence B (B.p 6068C). The mixture was vacuum filtered and the product was washed with essence B to obtain 34 g of bis 3.3 5.5-ethylenedioxy-l lB-allyl-4.5- seco-estrane-9o1, 17B-diol melting at 140C. STEP C: l 1B-allyl-A- -estradiene-l7,8-ol-3-one 34 g of bis 3.3 5.5-ethylenedioxy-l1B-allyl-4.5- secoestrane-9oz, 17,8-diol were added with stirring to a mixture of 340 ml of acetone and 68 ml of N sulfuric acid and the mixture was stirred for 4 hours at room temperature. Then 500 ml of water were added and the mixture was extracted with methylene chloride to obtain 38 g of a product which was used as is. Nitrogen was bubbled through the 38 g of product in 760 ml of anhydrous methanol with stirring and the solution was cooled while bubbling nitrogen therethrough. 85 g of potassium hydroxide were added at a temperature below 20C and the mixture was allowed to stand 4 hours at room temperature while bubbling nitrogen therethrough. 100 ml of acetic acid were added thereto and the mixture was diluted with water and extracted with methylene chloride to obtain 25 g of product which was chromatographed to obtain llfi-allyl-N- estradiene-l 7B-ol-3-one.

STEP D: llfi-allybA "-estratriene-3.l7,8-diol 25 g of llB allylA-"-estradiene-17,8-01-3-one were added with stirring under a nitrogen atmosphere to 125 ml of acetic acid anhydride and the solution was heated to 100C for 2 hours and was then cooled. 62.5 ml of acetyl bromide were added dropwise at a temperature below 10C and the mixture was allowed to stand for 2 hours at C and was then poured into ice. The mixture was extracted with methylene chloride and the extracts were washed with water. dried and distilled to dryness to obtain 36 g of a product which was chromatographed over silica to obtain 27 g of a resin used The 27 g of resin were dissolved at C under a nitrogen atmosphere in 540 ml of methanol and 27 ml of sodium carbonate were added thereto. The mixture stood at room temperature for 4 hours and then 16 ml of acetic acid and 2.5 liters of water were added thereto. The mixture was vacuum filtered and the precipitate was washed to obtain 19.65 g of product melting at 195C. The product was empasted at reflux with dichloroethane to obtain 1lB-allyl-A" -estratriene- 3,17B-diol melting at 200C.

STEP E: 3,7-dibenzoate of 1'1,B-allyl-A -estratriene-3,17B-diol V 9.3 ml of benzoyl chloride were added to a stirred 5 mixture of 9.286 g of llB-allyl-A" -estratriene- 3,17B-diol in 93 ml of anhydrous pyridine and the mixture was allowed to stand for 4 hours at room temperature. Water was then added to obtain a final volume of dilution of 250 ml and the mixture was vacuum filtered. I The precipitate waswashed with water and dried to ob estratriene3,17/3-diol melting at 198C. STEP F: 3.17-dibenzoate of 11B-[2'-0xoethyl]- A -estratriene-3,17B-diol a nitrogen atmosphere to 370 mlof methylene chloride and after cooling the solution to -65C, ozonized oxy- 3 gen was bubbled therethrough at'a rate of 0.2-0.3 liters per minute for 1V2 hours. Then, 25 g ofpowderedzinc and 50 ml of acetic acid were added and the temperature was allowed to return to room temperature. The

mixture was stirred for 30 minutes and the zinc was fil-,

tered off. The filtrate was washed, dried and distilled to graphed over silica gel to obtain 11.15 g of the 3,17

3.1734101 melting at 148C. STEP G: A" -estratriene-3. acid 1.2 g ofa 50% dispersion of sodium hydride in an oil were added with stirring under a nitrogen atmosphere to 20 ml of anhydrous tetrahydrofuran and after cool-I ing the mixture to 15C, 5 ml of methyl diethylphosphonoacetate were added .dropwise at atemperature below 20C. The mixture stood for a half hour and then 1.305 g'of 3,17-dibenzoate of llB-[2'oxoethyl]- A"r "-estratriene-3. l7B-diol in 20 ml of tetrahydrofuran was added dropwise. The mixture was stirred at extracts were washed with water, dried and distilled under reduced pressure to obtain 2.23 g of resin. The 2.23 g of resin wereadded to 20 ml of N sodium hy-- V droxide and 10 ml of ethanol and the mixture was re-' fluxecl for 1V2 hours and then cooled.'The mixture was extracted with chloroform and then ethylacetate. The

organicphases werewashed and dried and empasted with ether. The mixture was vacuum filtered and the crystals were washed to obtain 830 mg of A""- "-estratriene-3, 17B -diol-l lfi-crotonic acid melting at 272C.

acid

150 mg of 10% palladized carbon black were added 1 to a solution of 726 mg of A" -estratriene-3,' 17B- diol-l 1,8-crotonic acid in 30 ml of methanol and 2 m1 of chloroform and the mixture was stirred with hydrogen until saturation was reached. The catalyst was filtered I off and the filtrate was evaporated to obtain 750mg of I resin. The said resin was dissolved in 7.5 ml of methanol and 0.75 cm of potassium hydroxide and the solution was refluxed, acidified, diluted with water and ex- 4 tracted with ethyl acetate. The extract was washed with water, dried and distilled to dryness under reduced pressure to obtain 674 mg of crystalline productlwhich was crystallized 'to obtain 604 mg of A -estratain 12.6 g of the 3,17-dibenzoate of 11 B-ally1-A- 5 12.3 g of the 3.l7'-dibenzoate of 11 '-a11 1- A estratriene-3.l7B-diol were added with stirring under a.

dryness to obtain 12.89 g of resin which was chromato- 17,8-diol-l lB-crotonic room temperature for 40 minutes and water was added. V The mixture was extracted with ethylacetate and the triene-3, 17B-diol-11B-butyric acid melting at 262c.

EXAMPLE 2 A -estratriene-3-ol-l7one-1 1B-butyric acid A suspension of 2.1 g of A -estratriene-3, 17B- diol-l IB-butyric acid in 100 ml of acetone was cooled to C and 25 ml of a solution of 8 N chromic anhydride in dilute sulfuric acid were added. 400 ml of water were then added over 30 minutes and the mixture was vacuum filtered. The crystalline product was washed with water to obtain 1.386 g of a product melting at 240C. The mother liquors were extracted with ethyl acetate to obtain 0.5 80 g of-a product melting at 240C and the two crops were crystallized from isopropanol to obtain 770 g of A -estratriene-3-oll7-one-11B-butyric acid melting at 256C.

EXAMPLE 3 A" -estratriene-3ol-17-one-7a-butyric acid STEP A:

estrene-3-one 2.6 g of cuprous chloride were added to 575 ml of a solution of 0.62 M magnesium in 4-chlorobutanol. tetrahydropyranyl ether in tetrahydrofuran cooled to 30C under a nitrogen atmosphere and after stirring for minutes, a solution of 95 g of the acetate of 6- dehydro-l 9-nor-testosterone in 300 ml of tetrahydrofuran was added over 70 minutes. The solution was held for 1 hour at C and then ml of acetic acid and 300 ml of water were added. The mixture was extracted with ether and the organic phase was washed with water, dried and evaporated to dryness under reduced pressure to obtain 174 g of an oil which was addedto 350 ml of methanol and 88 ml of 0.5 N hydrochloric acid. The mixture was refluxed for 2 hours. cooled and diluted with water. The mixture was extracted with methylene chloride and the organic phase was washed, dried and distilled to dryness under reduced pressure to obtain 122 g of resin which was chromatographed. The product was subjected overnight at room temperature to the action of 2 volumes of acetic acid anhydride and 2 volumes of pyridine and the mixture was concen trated to dryness to obtain 7a-[-4'-acetoxybutyl]-l 7B- acetoxy-A -estrene-3-one melting at 1 13C. STEP B: 7a-[4-hydroxybutyl]-A-estrene-l7/3-01- 3-one 12 ml of 2 N methanolic potassium hydroxide were added under a nitrogen atmosphere to a solution of 5 g of the product of Step A in 10 ml of methanol and the mixture was neutralized with acetic acid in about 30 minutes and was diluted with water. The mixture was extracted with methylene chloride to obtain 4.3 g of 7a-[4-hydroxybutyl]-A -estrene17B'ol-3-one which was used as is for the next step. STEP C: A -estrene-3,17-dione-7oz-butyric acid A solution of 10 ml of 8 N chromic anhydride in dilute sulfuric acid was added with stirring at 0C to a solution of the product of Step B in 250 ml of acetone and the mixture was stirred for one-half hour. Methanol and water were added to the mixture and the acetone was removed by distillation under reduced pressure.

The mixture was iced. vacuum filtered and the precipitate was washed with water and dried to obtain 3.9 g of N-estrene-3.17-dione-7a-butyric acid melting at 250C after crystallization from ethanol at C. STEP D: A -estratriene-3-ol-l7-one-7a-butyric acid A solution of 19 g of 'monopotassium phosphate in 1450 ml of distilled water were added with stirring-and air introduction to 3 g of hyflosurpercel in 1500 of a solution buffered to a pH of7 by addition of N sodium hydroxide and sufficient distilled water was added to obtain a volume of 2800 ml. 3 g of A-estrene-3. l 7-dione- 7a-butyric acid were added thereto followed by 30 mg of menadione, 35 ml of methanol and 30 g of Arthrobacter acetonic powder. The suspension was held at 34C for 1 12 hours while adding over 65 hours 15 g of acetonic powder and 15 ml of methanol and over 96 hours 15 g of acetonic powder. At l 12 hours, the pH of the solution was adjusted to 14 by addition of concentrated sodium hydroxide and 500 ml of ethyl acetate were added. The mixture was filtered and the filtrate was acidified with concentrated hydrochloric acid and saturated with ammonium sulfate. The aqueous phase was decanted and extracted with ethyl acetate. The extracts were washed, dried and concentrated to-dryness and the insoluble fraction was washed with methanol and water. The filtrate was adjusted to a pH of 1 by addition of concentrated hydrochloric acid and was evaporated to dryness. The 2 dry extracts were purified by chromatography to obtain 1.92 g of A- -estratriene-3-ol-17-one-7a-butyric acid melting at 206C after crystallization from aqueous acetic acid.

lR Spectrum (chloroform): l7-Ketone C=0 at 1773 acid at l710"'", free OH at 1710"" and aromatic at 1613, 1589 and 1499""".

EXAMPLE 4 A" -estratriene-3,17B-diol-7a-butyric acid 100 mg of sodium borohydride were added with cooling and stirring to 900 mg of A "-estratriene-3-ol- 17-one-7a-butyric acid in 6 ml of 0.5 N sodium hydroxide and 1 ml of methanol under a nitrogen atmosphere and another 100 mg of sodium borohydride were added after 1 hour. The mixture was stirred for 30 minutes and 4 ml of 2 N hydrochloric acid were added. The mixture was diluted. extracted and washed. The extracts were dried and evaporated to dryness under reduced pressure and the residue was chromatographed to obtain 816 mg of A "-estratriene-3,l7B-diol-7otbutyric acid in the form of an amorphous product.

1R Spectrum: C=0 at 1706"" and aromatic at 1610. 1584 and 1503""? EXAMPLE 5 A- "-estratriene-3,16a,17,8-triol-7a-butyric acid STEP A: Ethyl A -estratriene-3-ol-17-one-7abutyrate Gaseous hydrochloric acid was bubbled'through 100 ml of A- -estratriene-3ol-17-one-7a-butyric acid in 3 ml of ethanol until saturation was reached and the mixture was refluxed for 1 hour. The mixture was distilled to dryness under reduced pressure and the residue was taken up in ethyl acetate. The solution was washed, dried and evaporated to dryness and the resulting resin was chromatographed to obtain 81 mg of ethyl A" -estratriene-3-ol-l7-one-7ct-butyrate in the form of an amorphous product.

STEP B: Ethyl 3,l7-diacetoxy-A" "-estratetraene- 7oz-butyrate 150 mg of p-toluene sulfonic acid monohydrate were added to a solution of 1.41 g of ethyl A -estratriene-3-ol-17-one-7a-butyrate in 30 ml of redistilled isopropenyl acetate and the mixture was refluxed and distilled for 8 hours while maintaining constant volume by addition of isopropenyl acetate. The reaction mixture was cooled to room temperature and a solution saturated with sodium bicarbonate was added thereto. The mixture was extracted with methylene chloride and the extracts were washed, dried and evaporated to dryness to obtain 1.1 g of ethyl 3.17B-diacetoxy- A" -estratetraene-7a-butyrate which was used as in for the next step.

STEP C: Ethyl 3,17,8-diacetoxy-l(mafia-epoxy A" -estratriene-7a-butyrate A drop of pyridine was added to a solution of 936 mg of the ethyl product of Step B in 10 ml of methylene chloride and after placing the mixture on an ice bath. 2.5 ml of methylene chloride solution of 1.2 M of hexafluoroacetone hydroperoxide were added over minutes. The temperature was raised to room temperature and the mixture was stirred under a nitrogen atmosphere for 2 /2 hours. The mixture was extracted with methylene chloride and the organic phase was washed. dried and concentrated to dryness to obtain 1 g of ethyl 3.1 7B-diacetoxy-l6ml7a-epoxy-A -estratriene- 7a-butyrate which was used as is in the next step. STEP D: Methyl A"- -estratriene-3,16a-diol- 17-one-7oz-butyrate 1 ml of 6 N sulfuric acid was added to a solution of l g of the ester of Step C in 10 ml of methanol and the mixture was stirred for minutes at room temperature and then refluxed for 2 hours. The mixture was cooled to room temperature and was diluted with water and extracted with ethyl acetate. The extracts were washed. dried and evaporated to dryness to obtain 882 mg of methyl A" "-estratriene-3. 16a-diol-17-one-7a butyrate which was used as is for the next step.

STEP E: A" -estratriene-3.16a,17B-triol'7abutyric acid 900 mg of sodium borohydride were added under a nitrogen atmosphere to a solution of 882 mg of the product of Step D in 10 ml of methanol and then 450 mg of sodium borohydride were added over 1% hours. The mixture was maintained at room temperature for 1 hour and then 1 ml of distilled water and 1 ml of potassium hydroxide were added thereto. The mixture was refluxed for 15 minutes. cooled to room temperature and diluted with water. The mixture was acidified with concentrated hydrochloric acid and extracted with ethyl acetate. The extracts were washed, dried and concentrated to dryness to obtain 795 mg of resin which was purified by chromatography to obtain 575 mg of A -estratriene-l16a,17B-triol-7a-butyric acid melting at 160170C and having a specific rotation [04],, +42 (c=l% in ethanol).

EXAMPLEo A"" -estratriene-3 .l 7B-diol-7cx-undeCan l STEP A: 17B-acetoxy-7a-( 1 l-acetoxyundecanyl) A- estrene-3-one A solution of 65 g of the tetrahydropyranyl ether of l l-bromo-undecanol in 200 ml of tetrahydrofuranwas added to 5g of magnesium turnings in 40 ml of tetrahyf I drofuran to obtain an 0.4 M solution of magnesium de-= rivative of the tetrahydropyranyl ether of ll-bromoand a solution of 16 g of the acetate of 6-dehydro-l.9- nor-testosterone in 150 ml of tetrahydrofuran was added over 5 hours. The resulting suspension was stirred for 1 hour at 30Cand 10 ml of acetic acid were added. The mixture was returned to room temperature and water was added. The mixture was decanted and extracted with ethyl ether and the extracts were washed, dried and concentrated to dryness under reduced pressure to obtain 44 g of a resin. The resin was dissolved in330 ml of methanol and ml of 0.5 N hy drochloric acid were added thereto. Th solutionwas refluxed for 1% hours and was then evaporated to dry-.

ness under reduced pressure. The residuewas taken up in water and the solution was extracted with methylene chloride and filtered. The filtrate was dried and evaporated to dryness to obtain 34 g of a resin which was pu-.

rified by chromatography to obtain 24 g of product; The latter was acetylated with 2 volumesof acetic acid anhydride and 2 volumes of pyridine overnightat room temperature followed by the usual treatment and puri- I 14.3 g of 17B-acetoxy-7a-(11- fication to obtain acetoxy undecanyl)-A -estrene-3-one.

UN. Spectrum(ethanol) Max. at 240 nm Elm Max. at 304 nm 3121:16500 mil 2 STEP C: 7a-(11-hydroxyundecanyl)A"?' -estratriene-3,17B-diol 21.5 g of lead tetraacetate were added under a nitrogen atmosphere to a solution of 14.3 g of the diacetate of Step B in 430 ml of acetic acidand the. solution was heated to C for 4 hours and was then distilled to dryness. The residue was taken up in water and the solution was extracted. The extracts were washed, dried.

and distilled to obtain 16.2 g of a brown resin which was dissolved in 500 ml of acetic acid. 80 g of potassium acetate were added to the solution and the mixture was refluxed overnight. Acetic acid was distilled off and the residue was taken up in water and ex tracted. The resin was chromatographed to obtain 5.6 I

of a product which was saponified by refluxing'in' 10 volumes of methanol and 1 volume of sodium hydro ide for 5 minutes. Chromatography gave 3.24 g of amorphous 7a-(l1-hydroxy undecanyl)-A "-estra- UV. Spectrum (ethanol):

lnflex. towards 220 nm E,,.,,."" 163 lnflex. towards 230 nm E...,,,"*' 114 Max. at 280 nm E 52 e 2300 lnflex. towards 287 nm E, 46 e 2050 STEP D: 3-benzoyloxy-7a-( 1 1-hydroxyundecany1)- A -estratriene-l713-01 A mixture of 3.2 g of the product of Step C in 15 ml ofN sodium hydroxide and 7 ml of acetone was cooled on an ice bath and 2.3 ml of benzoyl chloride were added dropwise with stirring. The mixture was stirred for 15 minutes and was then diluted with water and extracted. The residue of the organic phasewas chromatographed to obtain 2.64 of 3-benzoyloxy-7a-(11- hydroxyundecanyl)-A" -estratriene-17B-ol. STEP E: 3-benzoyloxy-A -estratriene-17-one-7ozundecanoic acid The product of Step D was dissolved in 130 ml of acetone with stirring and after cooling to 5C. 4.1 ml of an 8 N oxidizing reagent of Heilbron were added over minutes. After another 5 minutes of stirring, methanol was added and the mixture was diluted with water and concentrated under reduced pressure. The mixture was extracted with ethyl acetate and the extracts were washed, dried and concentrated to dryness to obtain 3-benzoyloxy-A "-estratriene-17-one-7aundecanoic acid. STEP F: A" -estratriene-3,17B-diol-7a-undecanoic acid 10 ml of 2 N sodium hydroxide were added to a solution of the product of Step E in 50 ml of methanol and after an hour, the solution was cooled on an ice bath. 1.5 g of sodium borohydride were added and the mixture was stirred for 1 hour and acidified to a pH of 1. The mixture was extracted and the extracts were washed, dried and evaporated to dryness. The residue was chromatographed over silica gel to obtain 1.61 g of A -estratriene-3,17B-diol-7a-undecanoic acid.

U.V. Spectrum (ethanol):

lnflex. towards 219 nm E 160 Inflex. towards 229 nm E 115 Max. at 280 nm 4 6 2100 lnflex. towards 286 nm E 43 EXAMPLE 7 18 of 7-carboxymethoxyimino-A -estratriene-3.17B diol.

LR. Spectrum: C=O at 1727" and aromatic at 1618. 1592 and 1506""'.

EXAMPLE 8 7-carboxymethoxyimio-A ""-estratriene-3-oll7-one 0.84 ml of an 8 N chromic anhydride in solution in dilute sulfuric acid (Heilbron Jones Solution) was added dropwise to a mixture of 2.44 g of 7-carboxymethoxyiminoA- '-estratriene-3.17B-diol in 122 ml of acetone cooled to 0C and after stirring at 0C for 30 minutes. another 0.84 ml of Heilbron Jones solution was added thereto. After 3 hours. another 0.41 ml of Heilbron Jones solution was added and the mixture was stirred for another 30 minutes. 2 ml of methanol and then 10 ml of an aqueous solution saturated with sodium bicarbonate were added dropwise to the mixture which was then vacuum filtered. The filtrate was washed with acetone and concentrated and then 200 ml of ethyl acetate were added. The organic phase was washed, dried and concentrated 'to dryness to obtain 2.135 g of a resin which was chromatographed to obtain 1.848 g of 7-carboxymethoxyimino-A"- "-estratriene-3-ol-l 7-one.

Analysis: C2nH23O5N: Calculated: "/(C 67.21. 7(H 6.48, 7zN 3.91; Found: 67.1 6.7 3.6.

EXAMPLE 9 4-carboxymethyl-2'-semicarbazone of A" -estratriene-3.17B-diol-7-one A mixture of 10.2ml of methanol. 1.8 g of potassium N-carboxyhydrazinoglycinate and 8.9 ml ofa methanol solution of 43 mg/ml of hydrochloric acid was formed with stirring under a nitrogen atmosphere and after heating the resulting suspension to reflux. 1.020 g of A" -estratriene-3 .17B-diol-7-one was added thereto. After refluxing for 3 hours. the mixture was cooled and vacuum filtered and the precipitate was washed to obtain 2 g of resin. The resin was added to 50 ml of a methylene chloride solution of diazomethane titrating 12.1 g per liter and the mixture was held at 0C for 1 hour and was vacuum filtered. The filtrate was evaporated to dryness to obtain 1.926 g of resin which was chromatographed to obtain 825 mg of product. The latter was dissolved in 8 cm of ethanol and 3 ml of N sodium hydroxide were added thereto. The reaction mixture stood at room temperature for 20 minutes and was iced and then 3 m1 of N hydrochloric acid and 5 ml of water were added. The mixture was vacuum filtered and the crystals were washed and dried to obtain 3.43 mg of 4-carboxymethyl-2'-semicarbazone of A" estratriene-3,17B-diol-7-one.

EXAMPLE 10 Triethylamine salt of 7-carboxymethoxyimino-A" '-estratriene-3.17B- diol 1 g of aminoxyacetic acid hemichlorohydrate was added to a mixture of 1 g of A" -estratriene-3,1 7B- diol-7-one (US. Pat. No. 2,418,603) in 10 ml of ethanol and the 8 ml of N sodium hydroxide were added to the suspension. The resulting solution stood at room temperature for 3 hours and then 8 ml of N hydrochloric acid were added. The mixture was diluted and extracted with ethyl acetate. The extracts were washed. dried and distilled to dryness to obtain 1.23 g ota resin with an IR Spectrum (ethanol) of C=O at l748""' and OH at 3607"" 0.5 ml of triethylamine, and 4 ml of methanol were added to a solution of 1.23 g of the said resin in 10 ml of ethyl acetate and the solution was concentrated by azeotropic entrainment of methanol while adding ethyl acetate for a constant volume. After the methanol was removed. 0.2 ml of triethylamine was added. The mixture was iced and vacuum filtered and the precipitate was washed to obtain 0.990 g of the triethylamine salt of 7-carboxymethoxyimino-A*' -estratriene-3.17B- diol melting at 195C and having a specific rotation [al =+2l (c=l7( in ethanol).

EXAMPLE A Conjugation of Beef serum albumin with A -estratriene-3-ol-l7-0ne-llB-butyric acid 0.23 ml of tri n-butylamine and 0.063 ml of isobutyl chloroformate were added to a mixture of 178 mg of A "estratriene-3-ol-l7-one-llB-butyric acid in 5 ml of dioxane cooled to 12C and the mixture was held at 12C for20 minutes. Then, after cooling the solution to C. a solution of 0.770 g of beef serum albumin in 22 ml of water was added thereto and after complete dissolution. 22 ml of dioxane and 0.74 ml of N sodium hydroxide were added at 0C. The solution stood at 0C for 4 hours and the mixture was subjected to membrane dialysis for 18 hours. The pH of the resulting solution was adjusted to 4.2 by addition of 2 N hydrochloric acid and the solution was iced for 68 hours at 20C. The ice was allowed to melt and the product was decanted. The precipitate was dissolvled in 50 ml of an iced 1% sodium bicarbonate solution and the solution was subjected to a second membrane dialysis for 48 hours. The resulting solution was extracted with chloroform and the extracted product was subjected to lyophilisation to obtain 720 mg of beef serum albumin conjugated with A" -estratriene-3-ol-l7-onel 1B- butyric acid.

U.V. Spectrum (ethanol):

1 =6 lnflex. towards 270 nm E 9 Max. at 281 nm E 11.8

EXAMPLE B U.V. Spectrum (ethanol lnflex. towards 270 nm Max. at 279 nm 8 xt-m H Various modifications of the products and processes of the invention may be made without departing from the spirit or scope thereof and it is to be understood that the invention is intended to be limited only as defined in the appended claimsv J a We claim:

1. A steroid of the formula and Y is selected from the group consisting of hydrogen and -OH and when R is (CH ),,COOH in which a is equal to 2n 3 and n is 0, l or 2, R is and when R is hydrogen. R is selected from the group 2' consisting of and =N-Z-(CH COOH, b is a number from 3 to 18.0 is a number from 1 to 8 and Z is'selected from the group consisting of O- and -NHCONH-. 2. A compound of claim 1 wherein R, is.(CH

),,COOH, a 2n 3, n is O, l or 2 and R is butyric acid and A" -estratriene-3-ol-l7-one l1B butyric acid.

4. A compound of claim and R is and b is 3 to 18.

5. A compound of claim 4 selected from the group.

consisting of A- -estratriene-3,l7B-diol-7a-butyric acid, N "-estratriene-3-ol-17-one 7a-butyric acid.

acid

and

acid.

6. A compound of claim 1 wherein R, is hydrogen and R is =NZ('CH .-COOH.

wherein X is selected from the group consistingof=0 l in which R ishydrogen 7. A compound of claim 6 which is selected from the reacting the latter with an acylating agent to form a group consisting of 7-carboxymethoxyimino-A-- compound of the formula estratriene-3.l7B-diol, 7-carboxymethoxyimino- A- -estratriene-3-ol-l7-one and 4-carboxymethyl- 5 C H2 2-semicarbazone of A"" "-estratriene-3,l7B-diol- 7-one. I I &2

8. A process for the preparation of a compound of I claim 2 comprising reacting a compound of the formula /i/ wherein L IS an acyl of an organic carboxylic acid. subjecting the latter to ozonolysis to form a compound of the formula wherein K and K are blocked keto in the form of a ketal and L is acyl of an organic carboxylic acid with an epoxidation agent followed by reaction with a saponification agent to form a compound of the formula y OLI K I OH 6 c L'O K 20 CH=O reacting the latter with an alkyl dialkylphosphonoacyreacting the latter with an allyl magnesium halide to l f h f la form a compound of the formula O CPI-CH R,s

l CH H if wherein R R and R are the same or different alkyls of l to 8 carbon atoms and n has the above definition 40 followed by reaction with a saponification agent to obtain a compound of the formula reacting the latter with a deketalization agent and then F n+l a cyclization agent to obtain a compound of the for- CH mula 2 OH and reacting the latter with a hydrogenation agent to obtain the compound of claim 2.

9. A process for the preparation of a compound of reacting the latter with an aromatization agent to form cla'm 4 comprising reacting a Steroid 0f the formula a compound of the formula CH=CH2 l CH2 H OL wherein L is acyl of an organic carboxylic acid with a compound of the formula HalMg(CH.,),,CH. ,OM

wherein Hal is bromine or chlorine and OM is a blocked hydroxy in ether form followed by reaction with an acid agent and then an acid of the formula AcOH where Ac is acyl of an organic carboxylic acid or a functional derivative thereof to obtain a compound of the formula (CH -CH reacting the latter with a saponification agent to obtain a compound of the formula CHZOH reacting the latter with an oxidation agent to form a Y compound of the formula and reacting the latter with a dehydrogenation agent to} I 4 form a compound of the formula I I 

1. A STEROID OF THE FORMULA
 2. A compound of claim 1 wherein R1 is -(CH2)a-COOH, a 2n + 3, n is 0, 1 or 2 and R2 is
 3. A compound of claim 2 selected from the group consisting of Delta 1,3,5(10)-estratriene-3,17 Beta -diol-11 Beta -butyric acid and Delta 1,3,5(10)-estratriene-3-ol-17-one-11 Beta -butyric acid.
 4. A compound of claim 1 in which R1 is hydrogen and R2 is
 5. A compound of claim 4 selected from the group consisting of Delta 1,3,5(10)-estratriene-3,17 Beta -diol-7 Alpha -butyric acid, Delta 1,3,5(10)-estratriene-3-ol-17-one-7 Alpha -butyric acid, Delta 1,3,5(10)-estratriene-3,16 Alpha ,17 Beta -triol-7 Alpha -butyric acid and Delta 1,3,5(10)-estratriene-3,17 Beta -diol-7 Alpha -undecanoic acid.
 6. A compound of claim 1 wherein R1 is hydrogen and R2 is NZ(CH2)c-COOH.
 7. A compound of claim 6 which is selected from the group consisting of 7-carboxymethoxyimino- Delta 1,3,5(10)-estratriene-3,17 Beta -diol, 7-carboxymethoxyimino- Delta 1,3,5(10)-estratriene-3-ol-17-one and 4''-carboxymethyl-2''-semicarbazone of Delta 1,3,5(10)-estratriene-3,17 Beta -diol-7-one.
 8. A process for the preparation of a compound of claim 2 comprising reacting a compound of the formula
 9. A process for the preparation of a compound of claim 4 comprising reacting a steroid of the formula 